Endostatin is a 20-kDa proteolytic fragment of collagen XVIII [O'Reilly M S, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277-285.]. It specifically inhibits the proliferation and migration of vascular endothelial cells, and induces the apoptosis of vascular endothelial cells [Yamaguchi N, Anand-Apte B, Lee M, et al. Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding. EMBO J. 1999; 18: 4414-4423]. Furthermore, endostatin efficiently inhibits angiogenesis and tumor growth with low toxicity and no drug resistance [Boehm T, Folkman J, Browder T, et al. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997; 390: 404-407,4; Herbst R S, Hess K R, Tran H T, et al. Phase I study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol 2002; 20: 3792-3803]. Treatment of endothelial cells with endostatin leads to a variety of downstream effects related to angiogenesis [Abdollahi A, Hahnfeldt P, Maercker C, et al. Endostatin's antiangiogenic signaling network. Mol Cell 2004; 13: 649-663].
Many proteins including integrins, glypicans, laminin, tropomyosin, caveolin, VEGFR-1, VEGFR-2, and MMP2 have been reported to interact with endostatin and may serve as potential endostatin receptors [Rehn M, Veikkola T, Kukk-Valdre E, et al. Interaction of endostatin with integrins implicated in angiogenesis. Proc Natl Acad Sci USA 2001; 98: 1024-1029; MacDonald N J, Shivers W Y, Narum D L, et al. Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin. J Biol Chem 2001; 276: 25190-25196; Karumanchi S A, Jha V, Ramchandran R, et al. Cell surface glypicans are low-affinity endostatin receptors. Mol Cell 2001; 7: 811-822; Sasaki T, Fukai N, Mann K, et al. Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J 1998; 17: 4249-4256; Kim Y M, Jang J W, Lee O H, et al. Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase. Cancer Res 2000; 60: 5410-5413]. Recently, our previous discovery disclosed that cell surface nucleolin functions as a receptor for endostatin [Shi H, Huang Y, Zhou H, et al. Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin. Blood 2007; 110: 2899-2906]. It specifically locates on angiogenic blood vessels and mediates the anti-angiogenic and anti-tumor activities of endostatin. Moreover, the expression of nucleolin on the cell surface of endothelial cells can be induced by the treatment of VEGF and extracellular matrix and is mediated by intracellular nonmuscle myosin heavy chain 9 [Huang Y, Shi H, Zhou H, et al. The angiogenic function of nucleolin is mediated by vascular endothelial growth factor and nonmuscle myosin. Blood 2006; 107: 3564-3571]. Besides, nucleolin was firstly identified as a major nucleolus protein and has been demonstrated to play a critical and fundamental role in cell growth and proliferation [Srivastava M, Pollard H B. Molecular dissection of nucleolin's role in growth and cell proliferation: new insights. FASEB J 1999; 13: 1911-1922]. It was reported that nucleolin can shuttle to cytoplasma and membrane from the nucleolus [Borer R A, Lehner C F, Eppenberger H M, et al. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 1989; 56: 379-390]. Nucleolin expressed on the cell surface is characterized as a biomarker of angiogenic endothelial cells [Christian S, Pilch J, Akerman M E, et al. Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. J Cell Biol 2003; 163: 871-878].
The lymphatic system plays important roles in maintaining fluid balance in the body and the pathogenesis of many diseases, including chronic inflammation and cancer [Swartz M A. The physiology of the lymphatic system. Adv Drug Deliv Rev 2001; 50: 3-20; Baluk P, Tammela T, Ator E, et al. Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J Clin Invest 2005; 115: 247-257; Cao Y. Opinion: emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis. Nat Rev Cancer 2005; 5: 735-743]. Lymphangiogenesis, the sprouting of new lymphatic vessels from the pre-existing lymphatic system, may provide a way to facilitate the dissemination of cancer cells to sentinel lymph nodes and further distant organs [Cao Y. Opinion: emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis. Nat Rev Cancer 2005; 5: 735-743; Gao P, Zhou G Y, Zhang Q H, et al. Lymphangiogenesis in gastric carcinoma correlates with prognosis. J Pathol 2009; 218: 192-200]. VEGF-C and D, the most potent pro-lymphangiogenic factors reported so far, are demonstrated to intensively stimulate lymphangiogenesis, and promote tumor lymphatic metastasis through their receptor VEGFR-3 [Jussila L, Alitalo K. Vascular growth factors and lymphangiogenesis. Physiol Rev 2002; 82: 673-700; Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J 1996; 15: 290-298; Royston D, Jackson D G. Mechanisms of lymphatic metastasis in human colorectal adenocarcinoma. J Pathol 2009; 217: 608-619]. Other pro-angiogenic factors including VEGF-A, platelet-derived growth factor BB (PDGF-BB), angiopoietin-1, hepatocyte growth factor (HGF) and insulin-like growth factor 1/2 (IGF1/2), have also been reported to stimulate lymphangiogenesis [Kajiya K, Hirakawa S, Ma B, et al. Hepatocyte growth factor promotes lymphatic vessel formation and function. EMBO J 2005; 24: 2885-2895; Tammela T, Saaristo A, Lohela M, et al. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood 2005; 105: 4642-4648; Bjorndahl M, Cao R, Nissen L J, et al. Insulin-like growth factors 1 and 2 induce lymphangiogenesis in vivo. Proc Natl Acad Sci USA 2005; 102: 15593-15598; Cursiefen C, Chen L, Borges L P, et al. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest 2004; 113: 1040-1050; Cao R, Bjorndahl M A, Religa P, et al. PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell 2004; 6: 333-345]. While, only alternatively spliced soluble VEGFR-2 has been reported as an endogenous selective lymphangiogenesis inhibitor so far [Albuquerque R J, Hayashi T, Cho W G, et al. Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth. Nat Med 2009; 15: 1023-1030].
Interestingly, Nakamura group firstly described that endostatin produced by adenovirus-infection in tumor cells inhibits lymph node metastasis [Fukumoto S, Morifuji M, Katakura Y, et al. Endostatin inhibits lymph node metastasis by a down-regulation of the vascular endothelial growth factor C expression in tumor cells. Clin Exp Metastasis 2005; 22: 31-38]. Similar results were observed by Heljasvaara's group in J4 transgenic mice overexpressing endostatin [Brideau G, Makinen M J, Elamaa H, et al. Endostatin overexpression inhibits lymphangiogenesis and lymph node metastasis in mice. Cancer Res 2007; 67: 11528-11535]. Both of these studies imply that the anti-lymphangiogenic effect of endostatin is due to down-regulation of VEGF-C, either by tumor cells or mast cells in the tumor microenvironment. Since it is well accepted that vascular endothelial cells are the direct target of endostatin, it is unclear whether endostatin can directly affect the lymphatic endothelial cells.
As detailed hereinafter, the inventors demonstrate that endostatin directly inhibits mouse lymphatic endothelial cells (mLECs) and lymphangiogenic vessels via cell surface nucleolin as its receptor on mLECs, but does not affect quiescent adult lymphatic vessels. Our discoveries provide a new method of screening the suitable objects for endostatin therapy to prevent tumor lymphangiogenesis and lymph node metastasis.